1. Field of the Invention
The present invention relates to a material for immobilization of biologically active substance, as well as to a method for immobilization of said substance using said material.
2. Description of the Prior Art
A biologically active substance (hereinafter referred to simply as "active substance", in some cases) such as protein, nucleic acid, oligopeptide, oligonucleotide or the like, immobilized on an insoluble material is useful because the immobilization enables the easy utilization of the activity of said substance. Examples of such utilization includes the industrial utilization of immobilized enzyme in the field of biochemistry or science, the immunological utilization of immobilized antibody or antigen, and the utilization of immobilized nucleic acid as diagnostic drug.
In this connection, various methods for immobilization of active substance were reported. For immobilization of, for example, an enzyme, there are methods such as the following.
(1) A method for immobilizing an enzyme on a material by chemical bonding using a crosslinking agent, a condensation agent or the like, for example, a diazo method, a peptide method, an alkylation method, a method using a crosslinking agent and a method using an Ugi reaction [Immobilized Enzyme, pp. 9-41 (1986), edited by Ichiro Chihata and published from Kodansha Scientific].
(2) A method for immobilizing an enzyme on a material by ionic bonding (Immobilized Enzyme, pp. 41-43).
(3) A method for immobilizing an enzyme on a material by physical adsorption (Immobilized Enzyme, pp. 43-45).
For immobilization of a nucleic acid, there are methods such as the following.
(1) A method for immobilizing a modification group-containing nucleic acid on a material by chemical bonding, for example, a method for bonding a nucleic acid having a thiol group at the 5' terminal, to a thiol group-containing bead-like material by disulfide bonding [P. J. R. Day, P. S. Flora, J. E. Fox, M. R. Walker, Biochem. J., Vol. 278, pp. 735-740 (1991)]. Other methods falling in this method (1) are described in, for example, Soren R. R., Mette R. L., Svend E. R., Anal. Biochem., Vol. 198, pp. 138-142 (1991); Jonathan N K., Joseph L. W., Joseph P. D., Rachel E. M., Mary C., Eugene L. B., Nucleic Acids Res., Vol. 15, pp. 2891-2909 (1987); Allan J. M., Jeffrey R. B., Terence W. P., Biochem J., Vol. 191, pp.276-279 (1990); and J. A. Running, M. S. Ureda, Biotechniques, Vol. 8, pp. 276-279 (1990).
(2) A method for immobilizing a nucleic acid on a material by physical adsorption, for example, a method for immobilizing a nucleic acid on a nitrocellulose or nylon film by physical adsorption using ultraviolet rays or heat (J. Sambrok, E. F. Fritsh, T. Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory Press, Second Edition, pp. 2.109-2.113 and pp. 9.36-9.46) and a method for immobilizing a nucleic acid on a microplate by physical adsorption [G. C. N. Parry, A. D. B. Malcolm, Biochem. Soc. Trans., Vol. 17, pp. 230-231 (1989)].
It is pointed out that the above conventional methods have problems. For example, in the methods by chemical bonding, special reagents are necessary and some of them (e.g. azide, isocyanate and NaBH.sub.3 CN) are poisonous. Moreover, the procedure of immobilization is complicated as follows. For example, when immobilization is conducted via a peptide bond, it is necessary to introduce an amino group into either one of the active substance and the material and a carboxyl group into the other and these two kinds of groups introduced must be reacted with each other for immobilization by the use of a condensation agent.
Also in the methods by chemical bonding, the material used must have a functional group (for example, when glutaraldehyde is used as a crosslinking agent, the material and the active substance each must have an amino group), and a material allowing for immobilization must be selected carefully. Further, a substance having no active group is impossible to immobilize on a material. For example, a substance (e.g. a natural DNA, or a synthetic DNA having no modification group) having only a functional group of low reactivity (e.g. terminal phosphoric acid group or terminal hydroxyl group) is difficult to immobilize by the method of chemical bonding.
Meanwhile in the methods by physical adsorption, there are problems such as (1) the amount of active substance immobilized varies depending upon the adsorbability of material, (2) the active substance adsorbed is desorbed easily, (3) when the active substance has a low molecular weight (oligomer), its interaction with the material is weak, making the adsorption difficult. Further, when a nucleic acid is immobilized on a nylon or nitrocellulose film by adsorption, both the adsorption density and the bonding strength are high but the film has insufficient strength and is broken easily, requiring thorough care in handling.